MiR-34 inhibits polycomb repressive complex 2 to modulate chaperone expression and promote healthy brain aging

Aging is a prominent risk factor for neurodegenerative disease. Defining gene expression mechanisms affecting healthy brain aging should lead to insight into genes that modulate susceptibility to disease. To define such mechanisms, we have pursued analysis of miR-34 mutants in Drosophila. The miR-34 mutant brain displays a gene expression profile of accelerated aging, and miR-34 upregulation is a potent suppressor of polyglutamine-induced neurodegeneration. We demonstrate that Pcl and Su(z)12, two components of polycomb repressive complex 2, (PRC2), are targets of miR-34, with implications for age-associated processes. Because PRC2 confers the repressive H3K27me3 mark, we hypothesize that miR-34 modulates PRC2 activity to relieve silencing of genes promoting healthful aging. Gene expression profiling of the brains of hypomorphic mutants in Enhancer of zeste (E(z)), the enzymatic methyltransferase component of PRC2, revealed a younger brain transcriptome profile and identified the small heat shock proteins as key genes reduced in expression with age.


Supplementary Figure 3: Independently integrated reporter transgenes confirm that Su(z)12 is a target of miR-34 in the brain (Associated with
) a, b. Su(z)12 protein levels are de-regulated in miR-34 mutants. Su(z)12 protein was detected using a transgene containing the Su(z)12 genomic region tagged with an HA epitope on the Cterminus of the predicted protein (Su(z)12-C-term HA). a. Western immunoblotting to detect Su(z)12 reporter levels in dissected brain. b. Quantification of Su(z)12 levels. Su(z)12 reporter expression decreased with age in wild-type, but is elevated in miR-34 mutants. Protein samples from dissected brains. Su(z)12-C-term HA protein levels were normalized to tubulin loading control. Mean ± SEM, n=4 biological repeats. Significant main effects were observed (genotype: F1,12=15.5, p<0.002 age: F1,12=43.9, p<0.001). (*p<0.05, **p<0.01, two-way ANOVA with Tukey post-test). c, d. The regulation of Su(z)12 protein levels by miR-34 is dependent upon the miR-34 seed sequence in the 3'UTR of the Su(z)12 transcript. The Su(z)12-C-term HA-mutant 3'UTR transgene contains a mutation in the miR-34 seed sequence that relieves the transcript from regulation by miR-34 (see Fig. 1d). c. The seed mutant is designed to disrupt base pairing between the Su(z)12 3'UTR of the transgene and miR-34. Su(z)12-C-term HA-mutant 3'UTR protein levels still decrease with age, but are no longer increased in miR-34 mutants, and are in fact decreased relative to wild-type. Protein samples from dissected brains. d. Quantification of Su(z)12-C-term HA-mutant 3'UTR protein levels using Western immunoblotting, normalized to tubulin loading control. Mean ± SEM, n=4 biological replicates. Significant main effects were observed (genotype: F1,12=40.4, p<0.001 age: F1,12=41.5, p<0..001). (*p<0.05, **p<0.01 two-way ANOVA with Tukey post-test).   12 4 . f. Young animals (3d) heterozygous for PRC2 mutations and expressing SCA3trQ78 have no significant developmental or overproliferation effects of photoreceptors. Each panel shows seven ommatidial units, with only mild photoreceptor cell loss induced by SCA3trQ78. Scale bar indicates 10µm. g. Histogram showing the distribution of photoreceptors (PR) per ommatidium at 3d in wild-type (black, 7.0 PR), SCA3trQ78 (red, 6.91 ± 0.08 SEM), SCA3trQ78; Su(z)12 2 /+ (blue, 6.92 ± 0.03 SEM), SCA3trQ78; Pcl 5 /+ (yellow, 6.98 ± 0.03 SEM), SCA3trQ78; E(z) 731 /+ (purple, 6.93 ± 0.07 SEM). n=10 flies for each genotype. Experimental design and biological replicates. Samples profiled were designed to assess changes in gene expression due to E(z) mutation at two separate temperatures to account for a presumptive temperature-sensitive allele, and to assess changes in gene expression due at advanced age. b. Principal component analysis of RNA-seq samples. Principal component analysis plot on rLog Normalized gene counts using DESeq2. Samples from similar genotype, temperature, and age cluster tightly together. c. Gene overlaps. Overlap between genes downregulated with age and upregulated in E(z). d. Gene overlaps between all categories of samples. Overlap between all regulated genes among both age and genotype contrasts. e. Genes negatively correlated with age show no bias towards upregulation or downregulation in E(z) mutants. Grey data points are genes not differentially expressed. Black data points are differentially expressed genes (p<0.05). Red circles are genes corresponding to probe sets previously defined as negatively correlated with age 1 . The overlap between genes called either upregulated or downregulated in E(z) mutants and the genes negatively-correlated with age is not statistically significant (hypergeometric test). f. Ordered list analysis of the genes negatively-correlated with age. Two ranked lists of genes were compared: a list of genes ranked by degree of upregulation in E(z) mutants (Supplementary Data 1) and the top 500 genes ranked by negative correlation with age 1 (Supplementary Data 2). E(z) upregulated genes were ranked by the product of the -log(p value) and the moderated log2 fold-change (high to low), such that genes upregulated in E(z) mutants are at the top. All negatively age-correlated genes were ranked according to increasing p value of the correlation with age among all genes with a negative correlation, followed by genes with positive correlation which were ranked according to decreasing p value. Yellow bars indicate 95% confidence intervals. There was no statistically significant overlap. g. Small HSPs are upregulated in E(z) mutant brains. Regulation of mRNA levels of CG7409, Hsp23, Hsp26, Hsp67Ba, and Hsp67Bc by E(z) was confirmed using qPCR on cDNA prepared from the samples used in RNA-seq. For each gene, the levels are elevated in E(z) mutant brains. Asterisks indicate significance of main effect of genotype in two-way ANOVA (**p<0.01, *p<0.05). CG7409: F1,12=20.9. Hsp23: F1,12=11.9. Hsp26: F1,12=18.7. Hsp67Ba: F1,12=6.1. Hsp67Bc: F1,12=26.5. h. Hsp70 is upregulated in E(z) mutant brains. Regulation of mRNA levels of Hsp70 by E(z) was confirmed using realtime qPCR on cDNA prepared from the samples used in RNA-seq. Asterisks indicate significance of main effect of genotype in two-way ANOVA (F1,12=14.8 **p<.01). This assay detects three isoforms of Hsp70 in addition to Hsp70Ba, which is upregulated 10-fold in E(z) mutants in the RNA-seq data.

Supplementary Table 1: Pcl and Su(z)12 are predicted targets of miR-34
Tabulation of microRNA prediction results from several algorithms. Four prediction algorithms were used to assess whether miR-34 targets Pcl and Su(z)12 in Drosophila, SUZ12 and PHF19 in mouse, and SUZ12, PHF19, and MTF2 in humans. "-" indicates that the algorithm does not predict the given gene as a target, and a "+" indicates that it does. Programs used were DIANA 2 ; Miranda 3 ; Pictar 4 ; Targetscan 5 . No target sites were predicted using these programs in SUZ12 or MTF2.

Species
Gene TargetSCAN Table 2: DAVID analysis of differentially regulated gene sets in brains of E(z) and in the brain with age GO Terms, KEGG Pathways, and InterPro Motifs associated with genes called downregulated in E(z) mutant brains, genes upregulated with age, genes upregulated in E(z) mutant brains, and genes downregulated with age. The "Category" of each gene set is indicated on the left, "Term" is the name of the given gene set, "Gene Count" is the number of genes called differentially expressed in the given gene set, with the percentage indicated in parentheses. On the right is the Benjamini adjusted p value, a threshold for which was set at 0.1.

Supplementary Table 4: Genotyping primers
Genotyping primers indicates primers used to genotype the E(z) 61 and E(z) 731 alleles in a heterozygous background. Column "Allele" indicates the allele being genotyped. Column "L Primer" and "R Primer" indicate the sequence of the primers used in PCR. Column "enzyme" indicates the restriction endonuclease used to resolve the different alleles. Column "+/+ (bp)" indicates the restriction fragment size expected for wild-type DNA amplified with the given primers and digested with the given enzyme. Column "+/-(bp)" indicates the sized expected if the DNA amplified was from a heterozygous animal.   Antibodies and experimental parameters used in western immunoblotting. Pcl and Histones are high pI proteins that required a higher pH transfer buffer. The first few columns list the target, catalog number, Lot, and manufacturer of each antibody used. Column "Pre-Blocking" indicates the blocking buffer used. Column "Gel type" indicates the various gels used for each antigen.
Column "Transfer Type / Buffer" indicates the methods used to transfer the proteins to PVDF membrane. Column "Transfer time & temperature" indicates transfer conditions. Column "1° dilution" indicates the concentration of primary antibody used relative to the manufacturer supplied stock. Column "primary antibody buffer" indicates the buffer used in primary antibody incubations. Columns "2° catalog number", "2° antibody dilution", and "2° antibody buffer" indicate the parameters used for secondary antibody incubation. After each antibody incubation, the immunoblots were rinsed as indicated in Column "Post-incubation Rinses"